Phosphorus is an essential element for the growth of all organisms. In livestock production, feed must be supplemented with inorganic phosphorus in order to obtain a good growth performance of monogastric animals (for example, pigs, poultry and fish).
In contrast, no inorganic phosphate needs to be added to the feedstuffs of ruminant animals. Microorganisms, present in the rumen, produce enzymes which analyze the conversion of phytate (myo-inositolhexakis-phosphate) to inositol and inorganic phosphate.
Phytate occurs as a storage phosphorus source in virtually all feed substances originating from plants. Phytate comprises 1-3% of all nuts, cereals, legumes, oil seeds, spores and pollen. Complex salts of phytic acid are termed phytin. Phytic acid is considered to be an anti-nutritional factor since it chelates minerals such as calcium, zinc, magnesium, iron and may also react with proteins, thereby decreasing the bioavailability of protein and nutritionally important minerals.
Phytate phosphorous passes through the gastro-intestinal tract of monogastric animals and is excreted in the manure. Though some hydrolysis of phytate does occur in the colon, the thus-released inorganic phosphorus has no nutritional value since inorganic phosphorus is absorbed only in the small intestine. As a consequence, a significant amount of the nutritionally important phosphorus is not used by monogastric animals, despite its presence in the feed.
The excretion of phytate phosphorus in manure has further consequences. Intensive livestock production has increased enormously during the past decades. Consequently, the amount of manure produced has increased correspondingly and has caused environmental problems in various parts of the world. This is due, in part, to the accumulation of phosphate from manure in surface waters which has caused eutrophication. For other background information, see European Patent Application Publication No. 420 358.
Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8) are enzymes that hydrolyze phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate and are known to be valuable feed additives.
A phytase was first described in rice bran in 1907 [Suzuki et al., Bull. Coll. Agr. Tokyo Imp. Univ. 7, 495 (1907)] and phytases from Aspergillus species in 1911 [Dox and Golden, J. Biol. Chem. 10, 183-186 (1911)]. Phytases have also been found in wheat bran, plant seeds, animal intestines and in microorganisms [Howsen and Davis, Enzyme Microb. Technol. 5, 377-382 (1983), Lambrechts et al., Biotech. Lett. 14, 61-66 (1992), Shieh and Ware, Appl. Microbiol. 16, 1348-1351 (1968)].
The cloning and expression of the phytase from Aspergillus niger (ficuum) has been described by Van Hartingsveldt et al., in Gene, 127, 87-94 (1993) and in European Patent Application, Publication No. 420 358 and from Aspergillus niger var awamori by Piddington et al. in Gene 133, 55-62 (1993).
Since phytases used so far in agriculture have certain disadvantages, it is an object of the present invention to provide new phytases or polypeptides having phytase activity with improved properties. Since it is known that phytases used so far lose activity during feed pelleting process due to heat treatment, improved heat tolerance would be such an improved property.
So far, phytases have not been reported in thermotolerant fungus with the exception of Aspergillus fumigatus [Dox and Golden et al., J. Biol. Chem. 10, 183-186 (1911)] and Rhizopus oryzae [Howson and Davies, Enzyme Microb. Technol. 5, 377-382 (1993)]. Thermotolerant phytases have been described originating from Aspergillus terreus Strain 9A-1 [Temperature optimum 70.degree. C.; Yamada et al., Agr. Biol. Chem. 32, 1275-1282 (1968)] and Schwanniomyces castellii [Temperature optimum 77.degree. C.; Segueilha et al., Bioeng. 74, 7-11 (1992)]. However for commercial use in agriculture such phytases must be available in large quantities. Accordingly it is an object of the present invention to provide DNA sequences coding for heat tolerant phytases. Improved heat tolerance of phytases encoded by such DNA sequences can be determined by assays known in the art, for example, by the processes used for feed pelleting or assays determining the heat dependence of the enzymatic activity itself as described, for example, by Yamada et al. (s.a.).
It is furthermore an object of the present invention to screen fungi which show a certain degree of thermotolerance for phytase production. Such screening can be made as described, for example, in Example 1. In this way heat tolerant fungal strains, listed in Example 1, have been identified for the first time to produce a phytase.
Heat tolerant fungal strains, see for example, those listed in Example 1, can then be grown as known in the art, for example, as indicated by their supplier, for example, the American Tissue Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Agricultural Research Service Culture Collection (NRRL) and the Centralbureau voor Schimmelcultures (CBS) from which such strains are available or as indicated, for example, in Example 2.
Further improved properties are, for example, an improved substrate specificity regarding phytic acid [myo-inositol (1,2,3,4,5,6) hexakisphosphate] which is a major storage form of phosphorous in plants and seeds. Since for the complete release of the six phosphate groups from phytic acid, a phytase and a pH 2.5. acid phosphatase activity are required, a polypeptide having phytase and pH 2.5 acid phosphatase activity would be highly desirable. For example, International. Patent Application Publication No. 94/03072 discloses an expression system which allows the expression of a mixture of phytate degrading enzymes in desired ratios. However, it would be even more desirable to have both such activities in a single polypeptide. Therefore it is also an object of the present invention to provide DNA sequences coding for such polypeptides. Phytase and phosphatase activities can be determined by assays known in the state of the art or described, for example, in Example 9.
Another improved property is, for example, a so called improved pH-profile. This means, for example, two phytin degrading activity maxima, for example, one at around pH 2.5 which could be the pH in the stomach of certain animals and another at around pH 5.5 which could be the pH after the stomach in certain animals. Such pH profile can be determined by assays known in the state of the art or described, for example, in Example 9. Accordingly it is also an object of the present invention to provide DNA sequences coding for such improved polypeptides.
It is yet another object of the present invention to provide a DNA sequence coding for a polypeptide having phytase activity and which DNA sequence is derived from a fungus selected from the group consisting of Acrophialophora levis, Aspergillus terreus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus sojae, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Humicola sp., Mycelia sterilia, Myrococcum thermophilum, Myceliophthora thermophila, Rhizomucor miehei, Sporotrichum cellulophilum, Sporotrichum thermophile, Scytalidium indonesicum and Talaromyces thermophilus or a DNA sequence coding for a fragment of such a polypeptide which fragment still has phytase activity, or more specifically such a DNA sequence wherein the fungus is selected from the group consisting of Acrophialophora levis, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Sporotrichum cellulophilum, Sporotrichum thermophile, Mycelia sterilia, Myceliophthora thermophila and Talaromyces thermophilus, or more specifically such a DNA sequence wherein the fungus is selected from the group consisting of Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Aspergillus nidulans and Talaromyces thermophilus. DNA sequences coding for a fragment of a polypeptide of the present invention can, for example, be between 1350 and 900, preferably between 900 and 450 and most preferably between 450 and 150 nucleotides long and can be prepared on the basis of the DNA sequence of the complete polypeptide by recombinant methods or by chemical synthesis with which one skilled in the art is familiar with.
Furthermore it is an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from the following:
(a) the DNA sequence of FIG. 1 [SEQ ID NO:1] or its complementary strand; PA1 (b) a DNA sequence which hybridizes under standard conditions with sequences defined under (a) or preferably with the coding region of such sequences or more preferably with a region between positions 491 to 1856 of such DNA sequences or even more preferably with a genomic probe obtained by preferably random priming using DNA of Aspergillus terreus 9A1 as described in Example 12. PA1 (c) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with sequences of (a) or (b), but which codes for polypeptides having exactly the same amino acid sequences as the polypeptides encoded by these DNA sequences; and PA1 (d) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b) or (c). PA1 (a) the DNA sequence of FIG. 2 [SEQ ID NO:3] or its complementary strand; PA1 (b) a DNA sequence which hybridizes under standard conditions with sequences defined under (a) or preferably a region which extends to about at least 80% of the coding region optionally comprising about between 100 to 150 nucleotides of the 5' end of the non-coding region of such DNA sequences or more preferably with a region between positions 2068 to 3478 of such DNA sequences or even more preferably with a genomic probe obtained by preferably random priming using DNA of Myceliophthora thermophila as described in Example 12. PA1 (c) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with sequences of (a) or (b), but which codes for polypeptides having exactly the same amino acid sequences as the polypeptides encoded by these DNA sequences; and PA1 (d) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b) or (c). PA1 (a) a DNA sequence comprising one of the DNA sequences of FIGS. 4 [SEQ ID NO:5], 5 [SEQ ID NO:7], 6 [SEQ ID NO:9] or 10 A and B ["aterr21", SEQ ID NO:13; "aterr58": SEQ ID NO:14] or its complementary strand; PA1 (b) a DNA sequence which hybridizes under standard conditions with sequences defined under (a) or preferably with such sequences comprising the DNA sequence of FIG. 4 [SEQ ID NO:5] isolatable from Talaromyces thermophilus, or of FIG. 5 [SEQ ID NO:7] isolatable from Aspergillus fumigatus, or of FIG. 6 [SEQ ID NO:9] isolatable from Aspergillus nidulans or of one or both of the sequences given in FIGS. 10A and B ["aterr21", SEQ ID NO:13; "aterr58": SEQ ID NO:14] isolatable from Aspergillus terreus (CBS 116.46) or more preferably with a region of such DNA sequences spanning at least 80% of the coding region or most preferably with a genomic probe obtained by random priming using DNA of Talaromyces thermophilus or Aspergillus fumigatus or Aspergillus nidulans or Aspergillus terreus (CBS 116.46) as described in Example 12; PA1 (c) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with sequences of (a) or (b) but which codes for polypeptides having exactly the same amino acid sequences as the polypeptides encoded by these DNA sequences; and PA1 (d) a DNA sequence which is a fragment of the DNA sequences specified in (a), (b) or (c). PA1 "ATGGA(C/T)ATGTG(C/T)TC(N)TT(C/T)GA" [SEQ ID NO:15] as sense primer and PA1 "TT(A/G)CC(A/G)GC(A/G)CC(G/A)TG(N)CC(A/G)TA" [SEQ ID NO: 16] as anti-sense primer. PA1 (a) "ATGGA(C/T)ATGTG(C/T)TC(N)TT(C/T)GA" [SEQ ID NO:15] as the sense primer and PA1 (b) "TA(C/T)GC(N)GA(C/T)TT(C/T)TC(N)CA(C/T)GA" [SEQ ID NO:17] as the sense primer and
"Standard conditions" for hybridization mean in this context the conditions which are generally used by one skilled in the art to detect specific hybridization signals and which are described, for example, by Sambrook et al., "Molecular Cloning" second edition, Cold Spring Harbor Laboratory Press 1989, New York, or preferably so called stringent hybridization and non-stringent washing conditions or more preferably so called stringent hybridization and stringent washing conditions one skilled in the art is familiar with and which are described, for example, in Sambrook et al. (s.a.) or even more preferred the stringent hybridization and non-stringent or stringent washing conditions as given in Example 12. "Fragment of the DNA sequences" means in this context a fragment which codes for a polypeptide still having phytase activity as specified above.
It is also an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from the following:
"Fragments" and "standard conditions" have the meaning as given above.
It is also an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from the following:
It is furthermore an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence is selected from a DNA sequence comprising the DNA sequence of FIG. 4 [SEQ ID NO:5] isolatable from Talaromyces thermophilus, of FIG. 5 [SEQ ID NO:7] isolatable from Aspergillus fumigatus, of FIG. 6 [SEQ ID NO:9] isolatable from Aspergillus nidulans or of FIGS. 10A and B ["aterr21": SEQ ID NO:13; "aterr58": SEQ ID NO:14] isolatable from Aspergillus terreus (CBS 116.46) or which DNA sequence is a degenerate variant or equivalent thereof.
"Fragments" and "standard conditions" have the meaning as given above. "Degenerate variant" means in this context a DNA sequence which because of the degeneracy of the genetic code has a different nucleotide sequence as the one referred to but codes for a polypeptide with the same amino acid sequence. "Equivalent" refers in this context to a DNA sequence which codes for polypeptides having phytase activity with an amino acid sequence which differs by deletion, substitution and/or addition of one or more amino acids, preferably up to 50, more preferably up to 20, even more preferably up to 10 or most preferably 5, 4, 3 or 2, from the amino acid sequence of the polypeptide encoded by the DNA sequence to which the equivalent sequence refers to. Amino acid substitutions which do not generally alter the specific activity are known in the state of the art and are described, for example, by H. Neurath and R. L. Hill in "The Proteins" (Academic Press, New York, 1979, see especially FIG. 6, page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well as these in reverse (the three letter abbreviations are used for amino acids and are standard and known in the art).
Such equivalents can be produced by methods known in the state of the art and described, for example, in Sambrook et al. (s.a.). Whether polypeptides encoded by such equivalent sequences still have a phytase activity can be determined by one of the assays known in the art or, for example, described in Example 9.
It is also an object of the present invention to provide one of the aforementioned DNA sequences which code for a polypeptide having phytase activity which DNA sequence is derived from a fungus, or more specifically such a fungus selected from one of the above mentioned specific groups of fungi.
Furthermore it is an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence hybridizes under standard conditions with a probe which is a product of a PCR reaction with DNA isolated from a fungus of one of the above mentioned groups of fungi and the following pair of PCR primer:
"Standard conditions" have the meaning given above. "Product of a PCR reaction" means preferably a product obtainable or more preferably as obtained by a reaction described in Example 12 referring back to Example 11.
Furthermore it is an object of the present invention to provide a DNA sequence which codes for a polypeptide having phytase activity and which DNA sequence hybridizes under standard conditions with a probe which is a product of a PCR reaction with DNA isolated from Aspergillus terreus (CBS 116.46) and the following two pairs of PCR primers:
"TT(A/G)CC(A/G)GC(A/G)CC(G/A)TG(N)CC(A/G)TA" [SEQ ID NO:16] as the anti-sense primer; and PA2 "CG(G/A)TC(G/A)TT(N)AC(N)AG(N)AC(N)C" [SEQ ID NO:18] as the anti-sense primer.
"Standard conditions" are as defined above and the term "product of a PCR reaction" means preferably a product obtainable or more preferably as obtained by a reaction described in Example 11.
It is furthermore an object of the present invention to provide a DNA sequence coding for a chimeric construct having phytase activity which chimeric construct comprises a fragment of a DNA sequence as specified above. The chimeric construct can comprise a fragment of a DNA sequence derived from a fungus. The fragment of a DNA sequence from a fungus can be fused to the fragment of another DNA sequence from another fungus. The N-terminal end of a DNA sequence from a fungus can be fused at its C-terminal end to the fragment of another DNA sequence from different fungus. The fungus from which the fragments can be selected include those from Acrophialophora levis, Aspergillus terreus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus sojae, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Humicola sp., Mycelia sterilia, Myrococcum thermophilum, Myceliophthora thermophila, Rhizomucor miehei, Sporotrichum cellulophilum, Sporotrichum thermophile, Scytalidium indonesicum and Talaromyces thermophilus, preferably selected from the group consisting of Acrophialophora levis, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Calcarisporiella thermophila, Chaetomium rectopilium, Corynascus thermophilus, Sporotrichum cellulophilum, Sporotrichum thermophile, Mycelia sterilia, Myceliophthora thermophila and Talaromyces thermophilus, more preferably selected from the group consisting of Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Aspergillus nidulans and Talaromyces thermophilus, and even more preferably such a DNA sequence wherein the chimeric construct consists at its N-terminal end of a fragment of the Aspergillus niger phytase fused at its C-terminal end to a fragment of the Aspergillus terreus phytase, or more preferably such a DNA sequence with the specific nucleotide sequence as shown in FIG. 7 [SEQ ID NO:11] and a degenerate variant or equivalent thereof, wherein "degenerate variant" and "equivalent" have the meanings as given above.
It is furthermore an object of the present invention to provide for the partial sequence of a 6 kb HindIII/KpnI insert of clone 1 (see FIG. 13, discussed herein) (SEQ ID NO:28), which includes the complete phytase-encoding gene of Aspergillus nidulans, a protein of 463 amino acids (SEQ ID NO:29).
It is furthermore an object of the present invention to provide for the partial sequences of a 5.5 kb EcoRI/SacI insert of clone Tt29-132 (see FIG. 17, discussed herein) (SEQ ID NO:30), which includes the complete phytase-encoding gene of Talaromyces thermophilus, a protein of 466 amino acids (SEQ ID NO:31).
It is furthermore an object of the present invention to provide for the partial sequence of a 6 kb BamHI fragment (see FIG. 19 discussed herein) (SEQ ID NO: 32), which included the complete phytase-encoding gene of Aspergillus fumigatus, a protein of 465 amino acids (SEQ ID NO:33).
It is furthermore an object of the present invention to provide for the partial sequence of a 2 kb KpnI insert of clone 227 (see FIG. 21 discussed herein) (SEQ ID NO:34), which includes the complete phytase-encoding gene of Aspergillus terreus (CBS116.46), a protein of 466 amino acids (SEQ ID NO:35).
Furthermore it is an object of the present invention to provide a DNA sequence as specified above wherein the encoded polypeptide is a phytase.
Furthermore, it is an object of the present invention to provide the polypeptides encoded by the above described DNA sequences which have phytase activity and fragments of the polypeptides which retain phytase activity, and in particular those polypeptides of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, and SEQ ID NO:35.
Genomic DNA or cDNA from fungal strains can be prepared as known in the art [see for example, Yelton et al., Procd. Natl. Acad. Sci. USA, 1470-1474 (1984) or Sambrook et al., s.a., or other standard reference for preparing cDNA from fungi] or, for example, as specifically described in Example 2.
The cloning of the DNA-sequences of the present invention from such genomic DNA can then be effected, for example, by using the well known polymerase chain reaction (PCR) method. The principles of this method are outlined for example, by White et al. (1989), whereas improved methods are described for example, in Innis et al. [PCR Protocols: A guide to Methods and Applications, Academic Press, Inc. (1990)]. PCR is an in vitro method for producing large amounts of a specific DNA of defined length and sequence from a mixture of different DNA-sequences. Thereby, PCR is based on the enzymatic amplification of the specific DNA fragment of interest which is flanked by two oligonucleotide primers which are specific for this sequence and which hybridize to the opposite strands of the target sequence. The primers are oriented with their 3' ends pointing toward each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences and extension of the annealed primers with a DNA polymerase result in the amplification of the segment between the PCR primers. Since the extension product of each primer can serve as a template for the other, each cycle essentially doubLes the amount of the DNA fragment produced in the previous cycle. By utilizing the thermostable Taq DNA polymerase, isolated from the thermophilic bacteria Thermus aquaticus, it has been possible to avoid denaturation of the polymerase which necessitated the addition of enzyme after each heat denaturation step. This development has led to the automation of PCR by a variety of simple temperature-cycling devices. In addition, the specificity of the amplification reaction is increased by allowing the use of higher temperatures for primer annealing and extension. The increased specificity improves the overall yield of amplified products by minimizing the competition by non-target fragments for enzyme and primers. In this way the specific sequence of interest is highly amplified and can be easily separated from the non-specific sequences by methods known in-the art, for example, by separation on an agarose gel and cloned by methods known in the art using vectors as described for example, by Holten and Graham in Nucleic Acid Res. 19, 1156 (1991), Kovalic et. al. in Nucleic Acid Res. 19, 4560 (1991), Marchuk et al. in Nucleic Acid Res. 19, 1154 (1991) or Mead et al. in Bio/Technolocy 9, 657-663 (1991).
The oligonucleotide primers used in the PCR procedure can be prepared as known in the art and described for example, in Sambrook et al. (1989 "Molecular cloning" 2nd edt., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
The specific primers used in the practice of the present invention have been designed as degenerate primers on the basis of DNA-sequence comparisons of known sequences of the Aspergillus niger phytase, the Aspergillus niger acid phosphatase, the Saccharomyces cerevisiae acid phosphatase and the Schizosaccharomyces pombe acid phosphat:ase (for sequence information see, for example, European Bioinformatics Institute (Hinxton Hall, Cambridge, GB). The degeneracy of the primers was reduced by selecting some codons according to a codon usage table of Aspergillus niger prepared on the basis of known sequences from Aspergillus niger. Furthermore it has been found that the amino acid at the C-terminal end of the amino acid sequences used to define the specific probes should be a conserved amino acid in all acid phosphatases including phytases specified above but the rest of the amino acids should be more phytase than phosphatase specific.
Such amplified DNA-sequences can than be used to screen DNA libraries of DNA of, for example, fungal origin by methods known in the art (Sambrook et al., s.a.) or as specifically described in Examples 5-7.
Once complete DNA-sequences of the present invention have been obtained they can be integrated into vectors by methods known in the art and described for example, in Sambrook et al. (s.a.) to overexpress the encoded polypeptide in appropriate host systems. However, one skilled in the art knows that also the DNA-sequences themselves can be used to transform the suitable host systems of the invention to get overexpression of the encoded polypeptide. Appropriate host systems are for example fungi, like Aspergilli, for example, Aspergillus niger [ATCC 9142] or Aspergillus ficuum [NRRL 3135] or like Trichoderma, for example, Trichoderma reesei or yeasts, like Saccharomyces, for example, Saccharomyces cerevisiae or Pichia, like Pichia pastoris, all available from ATCC. Bacteria which can be used are for example, E. coli, Bacilli as, for example, Bacillus subtilis or Streptomyces, for example, Streptomyces lividans (see for example, Anne and Mallaert in FEMS Microbiol. Letters 114, 121 (1993). E. coli, which could be used are E. coli K12 strains for example, M15 [described as DZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 (1974)], HB 101 [ATCC No. 33694] or E. coli SG13009 [Gottesman et al., J. Bacteriol. 148, 265-273 (1981)].
Vectors which can be used for expression in fungi are known in the art and described for example, in EP 420 358, or by Cullen et al. [Bio/Technology 5, 369-76 (1987)] or Ward in Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York (1991), Upshall et al. [Bio/Technology 5, 1301-1304 (1987)] (Gwynne et al. [Bio/Technology 5, 71-79 (1987)], Punt et al. [J. of Biotechnology 17, 19-34 (1991)] and for yeast by Sreekrishna et al. [J. Basic Microbiol. 28, 265-278 (1988), Biochem. 28, 4117-4125 (1989)], Hitzemann et al. [Nature 293, 717-722 (1981)] or in EP 183 070, EP 183 071, EP 248 227, EP 263 311. Suitable vectors which can be used for expression in E. coli are mentioned, for example, by Sambrook et al. [s.a.] or by Fiers et al. in Procd. 8th Int. Biotechnology Symposium" [Soc. Franc. de Microbiol., Paris (Durand et al., eds.), pp. 680-697 (1988)] or by Bujard et al. in Methods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc. Vol. 155, 416-433 (1987) and Stuber et al. in Immunological Methods, eds. Lefkovits and Pernis, Academic Press, Inc., Vol. IV, 121-152 (1990). Vectors which could be used for expression in Bacilli are known in the art and described, for example, in EP 405 370, Procd. Nat. Aciad. Sci. USA 81, 439 (1984) by Yansura and Henner, Meth. Enzym. 185, 199-228 (1990) or EP 207 459.
Either such vectors already carry regulatory elements, for example, promoters or the DNA-sequences of the present invention can be engineered to contain such elements. Suitable promotor-elements which can be used are known in the art and are, for example, for Trichoderma reesei the cbh1- [Haarki et al., Biotechnology 7, 596-600 (1989)] or the pki1-promotor [Schindler et al., Gene 130, 271-275 (1993)], for Aspergillus oryzae the amy-promotor [Christensen et al., Abstr. 19th Lunteren Lectures on Molecular Genetics F23 (1987), Christensen et al., Biotechnology 6, 1419-1422 (1988), Tada et al., Mol. Gen. Genet. 229, 301 (1991)], for Aspergillus niger the glaA- [Cullen et al., Bio/Technology 5, 369-376 (1987), Gwynne et al., Bio/Technlogy 5, 713-719 (1987), Ward in Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York, 83-106 (1991)], alcA- [Gwynne et al., Bio/Technology 5, 71-719 (1987)], suc1- [Boddy et al. Current Genetics 24, 60-66 (1993)], aphA- [MacRae et al., Gene 71, 339-348 (1988), MacRae et al., Gene 132, 193-198 (1993)], tpiA- [McKnight et al., Cell 46, 143-147 (1986), Upshall et al., Bio/Technology 5, 1301-1304 (1987)], gpdA- [Punt et al., Gene 69, 49-57 (1988), Punt et al., J. of Biotechnology 17, 19-37 (1991)] and the pkiA-promotor [de Graaff et al., Curr. Genet. 22, 21-27 (1992)]. Suitable promotor-elements which could be used for expression in yeast are known in the art and are, for example, the phos-promotor [Vogel et al., Molecular and Cellular Biology, 2050-2057 (1989); Rudolf and Hinnen, Proc. Natl. Acad. Sci. 84, 1340-1344 (1987)] or the gap-promotor for expression in Saccharamyces cerevisiae und for Pichia pastoris, for example, the aox1-promotor [Koutz et al. Yeast 5, 167-177 (1989); Sreekrishna et al., J. Basic Microbiol. 28, 265-278 (1988)].
Accordingly vectors comprising DNA sequences of the present invention, preferably for the expression of said DNA sequences in bacteria or a fungal or a yeast host and such transformed bacteria or fungal or yeast hosts are also an object of the present invention.
Once such DNA-sequences have been expressed in an appropriate host cell in a suitable medium the encoded phytase can be isolated either from the medium in the case the phytase is secreted into the medium or from the host organism in case such phytase is present intracellularly by methods known in the art of protein purification or described, for example, in EP 420 358. Accordingly a process for the preparation of a polypeptide of the present invention characterized in that transformed bacteria or a host cell as described above is cultured under suitable culture conditions and the polypeptide is recovered therefrom and a polypeptide when produced by such a process or a polypeptide encoded by a DNA sequence of the presert invention are also an object of the present invention.
Once obtained the polypeptides of the present invention can be characterized regarding their activity by assays known in the state of the art or as described, for example, by Engelen et al. [J. AOAC Intern. 77, 760-764 (1994)] or in Example 9. Regarding their properties which make the polypeptides of the present invention useful in agriculture any assay known in the art and described for example, by Simons et al. [British Journal of Nutrition 64, 525-540 (1990)], Schoner et al. [J. Anim. Physiol. a. Anim. Nutr. 66, 248-255 (1991)], Vogt [Arch. Geflugelk. 56, 93-98 (1992)], Jongbloed et al. [J. Anim. Sci., 70, 1159-1168 (1992)], Perney et al. [Poultry Science 72, 2106-2114 (1993)], Farrell et al., [J. Anim. Physiol. a. Anim. Nutr. 69, 278-283 (1993), Broz et al., [British Poultry Science 35, 273-280 (1994)] and Dungelhoef et al. [Animal Feed Science and Technology 49, 1-10 (1994)] can be used. Regarding their thermotolerance any assay known in the state of the art and described, for example, by Yamada et al. (s.a.), and regarding their pH and substrate specificity prcfiles any assays known in the state of the art and described, for example, in Example 9 or by Yamada et al., s.a., can be used.
In general the polypeptides of the present invention can be used without being limited to a specific field of application for the conversion of phytate to inositol and inorganic phosphate.
Furthermore the polypeptides of the present invention can be used in a process for the preparation of compound food or feeds wherein the components of such a composition, for example, feed and other nutrients, are mixed with one or more polypeptides of the present invention. The feed can then be fed to those animals, especially monogastic animals (for example, pigs and poultry). Accordingly compound food or feeds comprising one or more polypeptides of the present invention are also an object of the present invention. One skilled in the art is familiar with their process of preparation. Such compound foods or feeds can further comprise additives or components generally used for such purpose and known in the state of the art.
It is furthermore an object of the present invention to provide a method for the reduction of levels of phytate in animal manure characterized in that an animal is fed such a feed composition in an amount effective in converting phytate contained in the feedstuff to inositol and inorganic phosphate.